MicroRNA 374 as an Epigenetic Regulator of Chronic Pain

Abstract Chronic primary pain conditions (CPPCs) such as fibromyalgia and temporomandibular disorder (TMD) constitute a significant healthcare problem that affects over 100 million, predominately female, Americans. The origin of CPPCs is linked to genetic and environmental factors that enhance catecholamine tone. An estimated

2 in 3 patients with CPPCs have variants in the gene encoding catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines) that result in low COMT activity and increased catecholamine levels. Pain in these individuals is enhanced by stressful events (eg, motor vehicle collision) resulting in increased release of

catecholamines from sympathetic nerves. Our lab has shown that catecholamines drive pain via activation of peripheral beta-adrenergic receptor 3 (Adrb3) and downstream mediators that regulate neuronal excitability and immune responses. Catecholamine signaling has also been shown to alter the expression of microRNAs

(miRNAs), which are small non-coding RNAs that negatively regulate mRNA targets. However, the role of miRNA dysregulation in CPPC pathophysiology remains understudied and unclear. Preliminary data from our case- control study reveal that patients with TMD have decreased levels of miR-374. We replicated this finding in an

animal model of CPPCs where mice with low COMT activity exposed to stress exhibited pain and decreased levels of miR-374. In the same mice, 5 miR-374 mRNA targets that were dysregulated in patients with TMD (ATXN7, CRK, HIF1A, NUMB, and TGFBR2) were also dysregulated in adipose and spinal cord tissues, where

they are predicted to influence immune signaling and pain. HIF1A, NUMB, and TGFBR2 were upregulated in adipose from female mice, while ATXN7 and TGFBR2 were downregulated in spinal cord from male mice. These findings point to new RNA targets that may play an important role in pain related to heightened catecholamine

tone, yet mechanistic studies are needed to determine their causal role. Thus, the objective of this proposal is to directly test the relationship between miR-374, its mRNA targets, pain, and inflammation. My central hypothesis is that catecholamine activation of Adrb3 reduces levels of miR-374, leading to dysregulation of

mRNAs that promote inflammation and chronic pain in a sex hormone-dependent manner. I will use primary adipocytes and neurons to measure 1) miR-374 binding to mRNA targets using a luciferase reporter system and 2) the effects of Adrb3 activation and sex hormones on miR-374 and mRNA target expression using qPCR. In

our CPPC mouse model, I will also measure 3) the effects of synthetic miR-374 overexpression and antagonism on pain and cytokine production, and 4) miR-374 and mRNA target expression in distinct cell types using combined RNAscope and immunohistochemical methods. These results will elucidate our understanding of

epigenetic mechanisms contributing to CPPCs and identify novel targets for improved treatment options for those with these conditions. The proposed training plan will promote development of new in vitro and in vivo techniques and foster career advancement in a highly supportive and collaborative environment at Duke University.